Pathogen-free male Wistar rats (n=50; 8-week-old; 200 g) were obtained from the Institute of Drug Control (Qingdao, China). The rats were housed in individual cages in a temperature-controlled room with a 12-h light/dark cycle at 50–60% relative humidity and were given food and water ad libitum. The rats were allowed to acclimatize for 1 week prior to the dietary intervention. Protocols for the animal experiments were approved by the Qingdao University Animal Care and Use Committee (Shandong, China) and were developed in accordance with guidelines set by the National Institutes of Health Guide for Care and Use of Laboratory Animals. The present study was approved by the Ethics Committee of the Affiliated Hospital of Qingdao University.

The rat model of type 2 diabetes was generated according to a previously described method (33). The rats were randomly divided into two groups according to diet: Regular rat chow (n=10; normal control group) or high-calorie diet (n=40; 10% animal fat, 20% cane sugar, 2.5% cholesterol, 1% cholate and 66.5% regular chow). Following 8 weeks, rats fed the high-calorie diet were intraperitoneally injected with 30 mgxkg⁻¹ streptozocin to induce type 2 diabetes. One week later, fasting blood glucose and insulin levels were measured, and the insulin sensitivity index was calculated as 22.5/[fasting blood glucose (FBG) × insulin (INS)]. Rats with fasting blood glucose levels >10.0 mmol·l⁻¹ and higher insulin levels (n=40) were further subdivided into two groups: The ARB treatment group (n=20) receiving valsartan (40 mg/kgxday given orally; Novartis International AG, Basel, Switzerland), and the diabetes mellitus (DM) control group (n=20) receiving normal saline. These treatments were administered once a day for 12 weeks.

Following the 12-week treatment, the rats were weighed and urine samples were collected. The rats were then sacrificed, blood samples were obtained, and the kidneys were collected and weighed.

To evaluate albumin and creatinine excretion, 24-h urine samples were collected from the rats every 2 weeks during the 12-week treatment period. Albumin was measured using a turbidimetric immunoassay kit (Shibayagi Co., Ltd., Shibukawa, Japan). Creatinine was measured using an automatic biochemistry analyzer (model no. 7600-020; Hitachi, Ltd., Tokyo, Japan).

Blood samples were obtained from the tail vein once per week. FBG was determined using a glucometer (OneTouch™SureStep™; LifeScan, Inc., Milpitas, CA, USA). Serum insulin levels and glycated hemoglobin (HbA1c) levels were determined by enzyme-linked immunosorbent assay (cat. nos. YJ-58700 and YJ-0021a, Aquatic Diagnostic Ltd., Stirling, Scotland).

Serum creatinine, urea nitrogen, total cholesterol, triglyceride, low-density lipoprotein and high-density lipoprotein levels were determined using an automatic biochemistry analyzer. Creatinine clearance was calculated using the following formula: Creatinine clearance=urine creatinine concentration/[serum creatinine concentration × volume of urine (ml) per min)].

Blood pressure was measured using a tail cuff system (model LE5002; Diagnostic Systems Laboratories, Webster, TX, USA) in conscious rats, while animals rested in a climate-controlled room (23°C). Systolic blood pressure was measured five times consecutively.

Morphological characteristics of 50-nm renal cortex sections, including GBM thickness and the condition of podocyte processes, were examined and photographed using a JEM-1200 transmission electron microscope (JEOL, Ltd., Tokyo, Japan).

A conditionally immortalized mouse podocyte cell line, MPC5, was donated by The Central Laboratory of Shandong University (Shandong, China), and was cultured as previously described (34). The cell density at 60% were grown on plates in RPMI-1640 medium supplemented with 10% fetal bovine serum (cat. no. 1213G057, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), 10–50 U/ml recombinant mouse γ-interferon (IFN), 100 U/ml penicillin and 100 mg/ml streptomycin at 33°C under a humidified atmosphere containing 5% CO₂. Then the conditions were changed to 37°C (without γ-IFN) to induce cell differentiation into mature podocytes and cells were cultured for 10–14 days. Differentiated podocytes were then divided into five groups: Normal glucose (NG) group (5.6 mmol/l), high glucose (HG) group (30 mmol/l), high mannitol group (NG + mannitol 25 mmol/l), the valsartan (VAL) group (HG + the AngII receptor blocker VAR, 10⁻⁵ mmol/l), and HS group [HG + the TRP channel inhibitor (35), SAR7334, 1 µM) cultured in 37°C for 48 h.

Cultured podocytes or homogenized renal tissue were lysed in cold cell lysis buffer (50 mM Tris, 150 mM NaCl, 10 mM ethylene diamine tetraacetic acid and 1% Triton X-100) containing protease and phosphatase inhibitors. BCA protein assay kit (P0012, Beyotime Institute of Biotechnology, Haimen, China) and microplate reader (MD-SpectraMax, M5, USA) was used to determine total protein concentrations according to specification's instruction. The quantity of protein loaded per lane was 35 g/10 l. The proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently transferred to nitrocellulose membranes. Membranes were blocked with 3% nonfat dry milk for 2 h at room temperature. Primary antibodies against TRPC6 (sc-19196, goat anti-rat; 1:500), NFAT (sc-7296, mouse anti-rat; 1:2,000) and AngII (sc-20718, rabbit anti-rat; 1:1,000) were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) and incubated at 4°C overnight. Horseradish peroxidase-conjugated anti-immunoglobulin G served as the secondary antibody (ZB-2301, 1:5,000, Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.; OriGene Technologies, Inc., Beijing, China) and incubated at room temperature for 1 h, and proteins were detected using an enhanced chemiluminescence kit (Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.; OriGene Technologies, Inc.) Image J (version 1.8.0, National Institutes of Health, Bethesda, MD, USA) was used for intensity analysis.

Total RNA was isolated from kidney cortex samples and cultured podocytes using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The RNA was analyzed by 0.6% agarose gel electrophoresis, visualized by ethidium bromide and was quantified by spectrometry. Complementary cDNA was synthesized using PrimeScript RT Reagent Kit (cat. no. DRR037A; Takara Biotechnology Co., Ltd., Dalian, China). RT reactions were for 45 min at 25°C, 5 min at 85°C and then held at 4°C. RT-qPCR was carried out using SYBR Premix Ex Taq (Takara Biotechnology Co., Ltd.) on an ABI PRISM 7000 HT (cat. no. 11744-100; Applied Biosystems; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Sequences of primers targeting TRPC6, AngII, and NFAT2 are shown in Table I. Reactions were incubated at 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec, 60°C for 30 sec and 72°C for 40 sec, followed by melting curve analysis. Relative gene expression was performed using the comparative Cq method (2^−∆∆Cq) (36) with GAPDH as the internal control.

Results were expressed as the mean ± standard deviation of at least three independent experiments. Group differences were compared by one-way analysis of variance followed by Student-Newman-Keuls post hoc test. P<0.05 was considered to indicate a statistically significant difference. Least significant different t-test was used for pairwise comparison, while Tamhane's T2 was used for Heteroscedasticity. All analyses were performed using SPSS software (version 22; IBM Corp., Armonk, IL, USA).

The present study measured the metabolic parameters of rats in the normal control, DM and ARB treatment groups (n=10 per group). The results revealed that there was a lower insulin sensitivity index, and increased levels of FBG, HbA1c and lipids in the DM group than in the normal controls (Table II). However, fasting insulin and serum creatinine levels did not differ between the groups. In addition, no significant differences in metabolic parameters were observed between the DM and ARB treatment groups (Table II).

To assess glomerular injury, the present study measured urinary albumin excretion and found a significant difference between the DM and normal control groups. However, the ARB VAL significantly decreased urinary albumin excretion in the diabetic rats following 8 weeks of treatment (Fig. 1A). ARB treatment also decreased the elevated kidney weight/body weight ratio observed in diabetic rats (Fig. 1B) but did not affect creatinine clearance or systolic blood pressure (Fig. 1C and D).

To better understand the role of AngII in podocytes, the present study analyzed AngII expression changes in the glomerular podocytes of type 2 diabetic rats via western blotting and qPCR. In addition, the effects of ARB treatment on GBM thickness and podocyte effacement were examined by electron microscopy. In the DM group, the mRNA and protein levels of AngII were significantly higher than those of the normal control group (Fig. 2A and B); however, treatment with VAL (40 mg/kgxday for 12 weeks) decreased AngII expression to levels almost equivalent to those of the normal controls. In DM rats, diabetic nephropathy was observed, which manifested as GBM thickening and podocyte process effacement (Fig. 2C). In addition, impairment of the glomerular filtration barrier was ameliorated by ARB treatment (Fig. 2).

To determine whether TRPC6 and NFAT were involved in AngII-induced podocyte injury in type 2 diabetes, the present study evaluated the expression of TRPC6 and NFAT in the rat model employed. The results revealed higher mRNA and protein levels of TRPC6 and NFAT in diabetic rats when compared with the normal controls (Fig. 3). These changes were attenuated by ARB treatment (Fig. 3).

As the results of the present study thus far suggested that the effects of AngII in the pathogenesis of diabetic nephropathy were mediated by TRPC6/NFAT signaling, the present study then investigated whether this pathway was activated by HG levels in cultured podocytes. The protein levels (Fig. 4A-C) and mRNA levels (Fig. 4D-F) of AngII, TRPC6, and NFAT were markedly higher in cells exposed to HG (30 mmol/l) for 48 h when compared with cells exposed to NG levels (5.6 mmol/l). This effect was significantly attenuated by ARB treatment with VAL. Cells cultured with 5.6 mmol/l glucose and 25 mmol/l mannitol were also evaluated. The expression of AngII, TRPC6 and NFAT in these cells was similar to that of the NG control group, thereby ruling out the osmotic effect of glucose.

To further study the role of TRPC6 in this signaling pathway, the present study evaluated the effect of the TRP channel inhibitor SAR7334 on TRPC6 and NFAT expression in cultured podocytes. The results revealed that SAR7334 (the HS group) significantly attenuated the HG-induced increase of AngII, TRPC6 and NFAT expression, at the protein (Fig. 5A-C) and mRNA levels (Fig. 5D-F).